The present invention is the purification and cloning of chondroitin lyase enzymes found in Flavobacterium heparinum.
Glycosaminoglycans are unbranched polysaccharides consisting of alternating hexosamine and hexuronic residues which carry sulfate groups in different positions. This class of molecules can be divided into three families according to the composition of the disaccharide backbone. These are: heparin/heparan sulfate [HexA-GlcNAc(SO.sub.4)]; chondroitin sulfate [HexA-GalNAc]; and keratan sulfate [Gal-GlcNAc]. The chondroitin sulfate family includes seven sub-types designated unsulfated chondroitin sulfate, oversulfated chondroitin sulfate and chondroitin sulfates A-E which vary in the number and position of their sulfate functional groups. Additionally, chondroitin sulfate B, also referred to as dermatan sulfate, differs in that iduronic acid is the predominant residue in the alternative hexuronic acid position.
Chondroitin sulfates A, B and C are the predominant forms found in mammals and may be involved in the modulation of various biological activities including cell differentiation, adhesion, enzymatic pathways and hormone interactions. The presence of chondroitin sulfate proteoglycans is elevated in the later stages of cell growth in response to tissue and vessel damage, as reported by Yeo, et al., Am. J. Pathol. 138:1437-1450, 1991, Richardson and Hatton, Exp. Mol. Pathol. 58:77-95, 1993 and Forrester, et al., J. Am. Coll. Cardiol. 17:758-769, 1991. Chondroitin sulfates also have been associated with events involved in the progression of vascular disease and lipoprotein uptake as described by Tabas, et al., J. Biol. Chem., 268(27):20419-20432, 1993.
Chondroitin enzymes of a suitable purity and characterization could be useful tools in determining the role of chondroitin sulfates in modulating these cellular events and in developing therapeutics for the treatment of disease states.
Chondroitin sulfate degrading enzymes, referred to as chondroitinases or chondroitin sulfate lyases, from several bacterial species have been reported. Takegawa, et al., J. Ferm. Bioeng. 77(2):128-131, 1991, report a chondroitinase AC from Aureobacterium with a molecular weight of between 81,000 and 83,000 Daltons that is inhibited by copper ions. Bacteriodes thetaiotamicron produces two chondroitinase AC degrading enzymes of molecular weight 104,000 and 108,000 Daltons, as described by Linn, et al., J. Bacteriol. 165:859-866, 1985. Other bacterium including Flavobacterium heparinum, Proteus vulgaris, Arthrobacter aurescens and Pseudomonas fluorescens produce chondroitinase AC or chondroitinase ABC enzymes which are not well characterized, as reviewed by Linhardt, et al., Appl. Biochem. Biotechnol. 12:135-177, 1986. F. heparinum is the only microbe that produces an enzyme which is specific for dermatan sulfate, chondroitinase B, as reported by Linhardt, R., et al. However, the chondroitinase degrading enzymes from F. heparinum have not been purified to homogeneity or thoroughly characterized.
It is therefore an object of the present invention to provide methods for purifying chondroitin lyase enzymes.
It is a further object of the present invention to provide DNA sequences encoding chondroitin lyase enzymes.
It is a still further object of the present invention to provide purified chondroitin lyase enzymes which are useful as pharmaceutical regents.